Project Summary Human health is profoundly influenced by the relationship between humans and their commensal microbes. This delicately balanced relationship can be altered when microbial commensals evolve to become opportunistic pathogens; the virulence traits that allow this transition to occur are likely complex microbial adaptations. The evolutionary transition of commensal-to-pathogen is being studied in medically relevant bacterial species, but has received far less attention in fungal species. The molecular architecture and the evolutionary dynamics of the adaptations required for this transition are likely quite different in fungi than in bacteria, due to the complexity of eukaryotic cells and the likelihood of sexual reproduction in fungal microbes. The most abundant fungal commensal in humans, the yeast Candida albicans, is known to cause a wide range of opportunistic infections. Pathogenic strains are capable of forming biofilms? complex communities that are anchored to a surface and have increased resistance to antifungal therapies. These yeast biofilms form on a variety of surfaces, including medical implants, catheters, and in soft tissue; they are responsible for many resistant, chronic infections. Recent work has shown that the closely related model yeast, Saccharomyces cerevisiae, can also form biofilms; the traits associated with biofilm formation are just beginning to be studied. Given the availability of cellular, molecular, and genetic tools available for S. cerevisiae, and its status as an occasional opportunistic pathogen, it is the ideal organism to begin to explore the evolutionary transition from fungal commensal to pathogen. Using this model has the further benefit of being able to design the experiments in such a way that they can also address fundamental questions in evolution related to the molecular evolution of complex adaptations. In the proposed research, we will first use bulk segregant analysis with pooled sequencing (BSA-seq) to determine the genetic architecture of liquid biofilm formation in multiple natural isolates (Aim 1). This research will explore the amount of segregating variation that exists for this trait, as well as the extent of the influence of genetic background on the trait. Next, using the same natural isolates, we will evolve replicate asexual, sexual, and control populations for 1,000 generations under strong selection for plastic adherence and liquid biofilm formation (Aim 2). The populations will subsequently be sequenced at multiple time points in order to characterize the number of loci, the identity of alleles, the order of fixation, and the time required for this complex trait to evolve. A comparison of the sexual populations to the asexual populations will reveal the role of standing genetic variation and new mutational input. Finally, the results of Aim 1 will be compared to the results of Aim 2 in order to test whether the alleles identified in a QTL analysis can predict the outcome of selection (Aim 3). The ability of QTL mapping approaches to identify evolutionarily relevant information remains an open and fundamental question in evolutionary genetics.